Wastewater biotreatment is a cost effective, environmentally benign technology that is widely used by municipalities and industry to treat municipal waste or process waste. A variety of different processes that use microbes to remove inorganic and organic chemicals from industrial wastewater are known to those skilled in the art. For example, the activated sludge process is one common method. An activated sludge system usually involves a continuous flow process in which wastewater is mixed with sludge and aerated (Bitton, G. 1994. Wastewater Microbiology. Wiley-Liss, New York). The key feature of an activated sludge system is that some sludge is recycled from a settling tank back into the main reactor. The sludge is composed of bacteria and other microorganisms that utilize organic and inorganic chemicals in the wastewater as sources of nutrients and energy for growth. By utilizing the chemicals in the wastewater for metabolism and growth, the microorganisms incorporate the chemicals into new microorganisms and/or convert the chemicals into gases such as carbon dioxide and nitrogen, thereby removing the chemicals from the wastewater. Activation of sludge through recycling maintains a large population of microbes in the main reactor vessel to degrade the waste chemicals.
In general, a large variety of different types of bacteria may be found in a wastewater bioreactor. Bacteria belonging to the following genera are some of the bacteria that are likely to be present in a wastewater bioreactor: Acinetobacter, Bacillus, Brevibacterium, Comomonas, Flavobacterium, Pseudomonas, and Zooglea (Bitton, G. 1994. supra).The performance of a wastewater bioreactor is determined in part by the types of bacteria in the bioreactor because each bacterium growing in the bioreactor must have the right genes encoding biochemical pathways to use at least some of the different organic and inorganic chemicals available in the wastewater for energy and nutrients.
One example of a biochemical pathway that allows bacteria to use organic chemicals that might be in wastewater involves aromatic compounds such as toluene which are commonly found in industrial wastewater. The well characterized TOL plasmid pWWO contains xyl genes that encode enzymes for metabolism of toluene. These genes are organized into two operons (Assinder and Williams, Adv. Microb. Physiol. 31:2–69 (1990)). The upper pathway operon encodes the enzymes for oxidation of toluene to benzoate. The lower pathway operon encodes the 1,2-dioxygenase and benzoate dihydrodiol dehydrogenase that convert benzoate into catechol, the 2,3-catechol oxygenase that opens the aromatic ring of catechol, and the enzymes that then oxidize the resulting 2-hydroxy-muconic semialdehyde to intermediates of the TCA cycle. The TOL pathway typifies the general strategy that is used by many different bacteria to degrade a large variety of other aromatic compounds (Williams and Sayers, Biodegradation 5:195–217 (1994)), i.e., the aromatic compound is first converted to catechol or a substituted catechol, the aromatic ring of the catechol is opened in the second stage, and finally in the third stage of degradation, the ring cleavage product is converted to small aliphatic compounds that enter central metabolism. Hence, an influent containing aromatic compounds will favor growth of bacteria in the bioreactor that have the TOL pathway and/or funtionally similar pathways.
A bioreactor can be designed and operated to take advantage of different microbial activities. For example, the activated sludge process can be modified to encourage denitrification for removal of nitrate from wastewater (Bitton, G. 1994. supra). Denitrification is a process that involves anaerobic respiration during which nitrate serves as an electron acceptor in place of oxygen. During the course of denitrification, nitrate is reduced stepwise to elemental nitrogen (NO3→NO2→NO→N2O→N2) which is released to the atmosphere because of low water solubility. Optimum denitrification requires anoxic conditions because O2 represses denitrification. The most widespread genra containing denitrifying bacteria are probably Pseudomonas and Alcaligenes but other genera containing denitrifiers are common (Bitton, G. 1994. supra).
It is evident from the foregoing discussion that successful wastewater biotreatment with an activated sludge process and other biological processes requires that the appropriate microbes be present in the bioreactor. Optimum performance of the bioreactor involves monitoring and adjusting physical parameters such as pH and dissolved oxygen to maintain an appropriate environment for microbial metabolism. Failure of a wastewater bioreactor, as indicated by unacceptably high levels of chemicals in the effluent water, could result from a variety of problems, including loss of necessary microorganisms from the biotreatment system. Accordingly, routine monitoring of the types of microorganisms in a wastewater bioreactor would be useful in evaluating bioreactor performance and for anticipating system failures.
Rapid and accurate identification is essential for routine monitoring of wastewater bioreactor microorganisms. Traditional methods of microbial identification involve culturing the organism to be identified and performing standard tests that reveal biochemical characteristics of the organism (Busse, H., J., Denner, E. B., Lubitz W. 1996. Classification and identification of bacteria: current approaches to an old problem. Overview of methods used in bacterial systematics. J. Biotechnol. 47(1):3–38). The results of the tests are used to search a database for an organism with the same characteristics. Such systems are not practical for routine monitoring of a wastewater bioreactor because of the large number of different isolates that would have to be cultured and tested daily.
Methods of microorganism identification that involve the use of DNA probes based on the sequences of ribosomal RNA (rRNA) molecules can be used to routinely test a sample for many different organisms rapidly and accurately (Busse, H., J., et al., supra); Muyzer, G., and N. B. Ramsing. 1996. Molecular methods to study the organization of microbial communities. Water Sci. Technol. 32:1–9). All cells contain ribosomes. Each ribosome is composed of three distinct rRNA molecules and a variety of protein molecules. In bacteria, the medium sized rRNA molecule, i.e., the 16S rRNA molecule, is particularly useful for identifying bacteria (Ward, D. M., M. M. Bateson, R. Weller, and A. L. Ruff-Roberts. 1992. Ribosomal RNA analysis of Microorganisms as they occur in nature. Adv. Microbial Ecol. 12:219–286; Woese, C. R. 1987. Bacterial Evolution. Microbiol. Rev. 51:221–271). The nucleotide sequence of the 16S rRNA molecule has conserved regions that are present in most if not all bacteria and variable regions that can be used to distinguish species and subspecies. Since a rRNA molecule is a direct gene product that results from transcription of a corresponding rRNA gene (rDNA), rDNA can be specifically and rapidly isolated from a particular microorganism or a mixture of microorganisms by using appropriate DNA primers and the polymerase chain reaction (PCR) to amplify the rDNA. The pattern of fragments resulting from cutting the PCR product with a set of restriction endonucleases can be used to identify the organism from which the rDNA was amplified (Busse, H. J., et al., supra). Alternatively, in situ hybridization techniques are known whereby fluorescent probes based on specific 16S rRNA sequences can be used to demonstrate the presence of specific bacteria in samples of sludge (Wagner, M., Amann, R., Lemmer, H., Schleifer, K. H. 1993. Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ. Microbiol. 59:1520–1525).
The problem to be overcome is to rapidly identify microorganisms that may be found in a wastewater bioreactor so that the presence or absence of those microorganisms in the wastewater bioreactor can be monitored. The Applicants have solved the stated problem by identifying some of the bacteria that are present in a wastewater bioreactor and by providing sets of nucleic acid sequences that are unique to various novel bacterial strains that were isolated from an industrial wastewater bioreactor. The present invention has utility in that the provided sets of nucleic acid sequences can be used to identify and monitor the corresponding bacterial strains in samples taken from a wastewater bioreactor or from any environment which might contain the specified bacteria.